scholarly journals Does protein A mirror image exist in solution? Outline of an experimental design aimed to detect it

2018 ◽  
Author(s):  
Osvaldo A. Martin ◽  
Yury Vorobjev ◽  
Harold A. Scheraga ◽  
Jorge A. Vila
Keyword(s):  
Experimental Design ◽  
Protein A ◽  
Mirror Image ◽  
Direct Application ◽  
Computational Simulations ◽  
Native Conformation ◽  
Native State ◽  
Theoretical Evidence ◽  
Image State ◽  
Unsolved Issue

ABSTRACTThere is abundant theoretical evidence indicating that a mirror image of ProteinA may occur during the protein folding process. However, as to whether such mirror image exists in solution is an unsolved issue. Here we provide outline of an experimental design aimed to detect the mirror image of Protein A in solution. The proposal is based on computational simulations indicating that the use of a mutant of protein A, namely 01 OH, could be used to detect the mirror image conformation in solution. Our results indicate that the native conformation of the protein A should have a pKa, for the 01 OH mutant, at r06.2, while the mirror image conformation should have a pKa close to ≈7.3. Naturally, if all the population is in the native state for the 01 OH mutant, the pKa should be ≈6.2, while, if all are in the mirror image state, it would be ≈7.3, and, if it is a mixture, the pKa should be larger than 6.2, presumably in proportion to the mirror population. In addition, evidence is provided indicating the tautomeric distribution of H1O must also change between the native and mirror conformations. Although this may not be completely relevant for the purpose of determining whether the protein A mirror image exists in solution, it could provide valuable information to validate the pKa findings. We hope this proposal will foster experimental work on this problem either by direct application of our proposed experimental design or serving as inspiration and motivation for other experiments.

scholarly journals Outline of an experimental design aimed to detect protein A mirror image in solution

2019 ◽  
Vol 1 ◽  
pp. e2
Author(s):  
Osvaldo A. Martin ◽  
Yury Vorobjev ◽  
Harold A. Scheraga ◽  
Jorge A. Vila
Keyword(s):  
Experimental Design ◽  
Protein A ◽  
Mirror Image ◽  
Direct Application ◽  
Computational Simulations ◽  
Native Conformation ◽  
Native State ◽  
Theoretical Evidence ◽  
Image State ◽  
Unsolved Issue

There is abundant theoretical evidence indicating that a mirror image of Protein A may occur during the protein folding process. However, as to whether such mirror image exists in solution is an unsolved issue. Here we provide outline of an experimental design aimed to detect the mirror image of Protein A in solution. The proposal is based on computational simulations indicating that the use of a mutant of protein A, namely Q10H, could be used to detect the mirror image conformation in solution. Our results indicate that the native conformation of the protein A should have a pKa, for the Q10H mutant, at ≈6.2, while the mirror-image conformation should have a pKa close to ≈7.3. Naturally, if all the population is in the native state for the Q10H mutant, the pKa should be ≈6.2, while, if all are in the mirror-image state, it would be ≈7.3, and, if it is a mixture, the pKa should be larger than 6.2, presumably in proportion to the mirror population. In addition, evidence is provided indicating the tautomeric distribution of H10 must also change between the native and mirror conformations. Although this may not be completely relevant for the purpose of determining whether the protein A mirror image exists in solution, it could provide valuable information to validate the pKa findings. We hope this proposal will foster experimental work on this problem either by direct application of our proposed experimental design or serving as inspiration and motivation for other experiments.

scholarly journals The ‘Relevant’ Stability of Proteins with Equilibrium Intermediates

2002 ◽  
Vol 2 ◽  
pp. 1209-1215 ◽  
Author(s):  
Javier Sancho ◽  
Marta Bueno ◽  
Luis A. Campos ◽  
Juan Fernandez-Recio ◽  
Maria Pilar Iran ◽  
...  
Keyword(s):  
Side Chain ◽  
Practical Issue ◽  
Single Chain ◽  
Native Conformation ◽  
Native State ◽  
Protein Activity ◽  
Overall Stability ◽  
Protein Stabilisation

Proteins perform many useful molecular tasks, and their biotechnological use continues to increase. As protein activity requires a stable native conformation, protein stabilisation is a major scientific and practical issue. Towards that end, many successful protein stabilisation strategies have been devised in recent years. In most cases, model proteins with a two-state folding equilibrium have been used to study and demonstrate protein stabilisation. Many proteins, however, display more complex folding equilibria where stable intermediates accumulate. Stabilising these proteins requires specifically stabilising the native state relative to the intermediates, as these are expected to lack activity. Here we discuss how to investigate the ‘relevant’ stability of proteins with equilibrium intermediates and propose a way to dissect the contribution of side chain interactions to the overall stability into the ‘relevant’ and ‘nonrelevant’ terms. Examples of this analysis performed on apoflavodoxin and in a single-chain mini antibody are presented.

scholarly journals Native Conformation and Canonical Disulfide Bond Formation Are Interlinked Properties of HIV-1 Env Glycoproteins

2015 ◽  
Vol 90 (6) ◽  
pp. 2884-2894 ◽  
Author(s):  
Eden P. Go ◽  
Albert Cupo ◽  
Rajesh Ringe ◽  
Pavel Pugach ◽  
John P. Moore ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Protein A ◽  
Native Conformation ◽  
Purification Method ◽  
Immunodeficiency Virus ◽  
Monomeric Gp120 ◽  
Env Protein ◽  
The Impact ◽  
Hiv 1

ABSTRACTWe investigated whether there is any association between a native-like conformation and the presence of only the canonical (i.e., native) disulfide bonds in the gp120 subunits of a soluble recombinant human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein. We used a mass spectrometry (MS)-based method to map the disulfide bonds present in nonnative uncleaved gp140 proteins and native-like SOSIP.664 trimers based on the BG505envgene. Our results show that uncleaved gp140 proteins were not homogeneous, in that substantial subpopulations (20 to 80%) contained aberrant disulfide bonds. In contrast, the gp120 subunits of the native-like SOSIP.664 trimer almost exclusively retained the canonical disulfide bond pattern. We also observed that the purification method could influence the proportion of an Env protein population that contained aberrant disulfide bonds. We infer that gp140 proteins may always contain a variable but substantial proportion of aberrant disulfide bonds but that the impact of this problem can be minimized via design and/or purification strategies that yield native-like trimers. The same factors may also be relevant to the production and purification of monomeric gp120 proteins that are free of aberrant disulfide bonds.IMPORTANCEIt is widely thought that a successful HIV-1 vaccine will include a recombinant form of the Env protein, a trimer located on the virion surface. To increase yield and simplify purification, Env proteins are often made in truncated, soluble forms. A consequence, however, can be the loss of the native conformation concomitant with the virion-associated trimer. Moreover, some soluble recombinant Env proteins contain aberrant disulfide bonds that are not expected to be present in the native trimer. To assess whether these observations are linked, to determine the extent of disulfide bond scrambling, and to understand why scrambling occurs, we determined the disulfide bond profiles of two soluble Env proteins with different designs that are being assessed as vaccine candidates. We found that uncleaved gp140 forms heterogeneous mixtures in which aberrant disulfide bonds abound. In contrast, BG505 SOSIP.664 trimers are more homogeneous, native-like entities that contain predominantly the native disulfide bond profile.

scholarly journals Efficient Refolding of Aggregation-prone Citrate Synthase by Polyol Osmolytes

2005 ◽  
Vol 280 (16) ◽  
pp. 15553-15560 ◽  
Author(s):  
Rajesh Mishra ◽  
Robert Seckler ◽  
Rajiv Bhat
Keyword(s):  
Small Molecules ◽  
Molecular Chaperones ◽  
Rational Design ◽  
Protein Production ◽  
Citrate Synthase ◽  
Protein A ◽  
Complete Recovery ◽  
Hydroxyl Groups ◽  
Native State

Efficient refolding of proteins and prevention of their aggregation during folding are of vital importance in recombinant protein production and in finding cures for several diseases. We have used citrate synthase (CS) as a model to understand the mechanism of aggregation during refolding and its prevention using several known structure-stabilizing cosolvent additives of the polyol series. Interestingly, no parallel correlation between the folding effect and the general stabilizing effect exerted by polyols was observed. Although increasing concentrations of polyols increased protein stability in general, the refolding yields for CS decreased at higher polyol concentrations, with erythritol reducing the folding yields at all concentrations tested. Among the various polyols used, glycerol was the most effective in enhancing the CS refolding yield, and a complete recovery of enzymatic activity was obtained at 7mglycerol and 10 μg/ml protein, a result superior to the action of the molecular chaperones GroEL and GroESin vitro. A good correlation between the refolding yields and the suppression of protein aggregation by glycerol was observed, with no aggregation detected at 7m. The polyols prevented the aggregation of CS depending on the number of hydroxyl groups in them. Stopped-flow fluorescence kinetics experiments suggested that polyols, including glycerol, act very early in the refolding process, as no fast and slow phases were detectable. The results conclusively demonstrate that both the thermodynamic and kinetic aspects are critical in the folding process and that all structure-stabilizing molecules need not always help in productive folding to the native state. These findings are important for the rational design of small molecules for efficient refolding of various aggregation-prone proteins of commercial and medical relevance.

scholarly journals Folding processes of the B domain of protein A to the native state observed in all-atom ab initio folding simulations

2008 ◽  
Vol 128 (23) ◽  
pp. 235105 ◽  
Author(s):  
Hongxing Lei ◽  
Chun Wu ◽  
Zhi-Xiang Wang ◽  
Yaoqi Zhou ◽  
Yong Duan
Keyword(s):  
Ab Initio ◽  
Protein A ◽  
Native State ◽  
Folding Simulations

scholarly journals An Epilepsy-Causing Mutation Leads to Co-Translational Misfolding

2020 ◽  
Author(s):  
Janire Urrutia ◽  
Alejandra Aguado ◽  
Carolina Gomis-Perez ◽  
Arantza Muguruza-Montero ◽  
Oscar R. Ballesteros ◽  
...  
Keyword(s):  
Structural Instability ◽  
Side Chain ◽  
Binding Assays ◽  
Native Conformation ◽  
Native State ◽  
Iq Motif ◽  
Epileptic Patients ◽  
Nascent Chain ◽  
Inherited Mutations

AbstractProtein folding to the native state is particularly relevant in human diseases where inherited mutations lead to structural instability, aggregation and degradation. In general, the amino acid sequence carries all the necessary information for the native conformation, but the vectorial nature of translation can determine the folding outcome. Calmodulin (CaM) recognizes the properly folded Calcium Responsive Domain (CRD) of Kv7.2 channels. Within the IQ motif (helix A), the W344R mutation found in epileptic patients has negligible consequences for the structure of the complex as monitored by multiple in vitro binding assays and molecular dynamic computations. In silico studies revealed two orientations of the side chain, which are differentially populated by WT and W344R variants. Binding to CaM is impaired when the mutated protein is produced in cellulo but not in vitro, suggesting that this mutation impedes proper folding during translation within the cell by forcing the nascent chain to follow a folding route that leads to a non-native configuration, and thereby generating non-functional ion channels that fail to traffic to proper neuronal compartments.

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